Identification and characterization of a cell membrane nucleic acid channel.
We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 +/- 0.4 picosiemens (pS) and reversal potential was 0.2 +/- 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ion-substitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using 32P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.
Hanss
B
B
Division of Nephrology, Box 1243, Mt. Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA. b_hanss@smtplink.mssm.edu
Leal-Pinto
E
E
Bruggeman
L A
LA
Copeland
T D
TD
Klotman
P E
PE
D000818:Animals; D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D007473:Ion Channels / Q000302:isolation & purification* / Q000502:physiology; D007668:Kidney / Q000378:metabolism*; D008051:Lipid Bilayers; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D008871:Microvilli / Q000737:chemistry*; D009841:Oligonucleotides / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004268:DNA-Binding Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009841:Oligonucleotides